Radiofluorinated compounds and their preparation

ABSTRACT

The present invention provides a process for [ 18 F]-fluorination of biomolecules containing a primary amino group such as proteins and peptides and in particular of peptides. The invention further provides reagents for this process, in particular  18 F-labelled prosthetic groups for use in the preparation as well as non-labelled intermediates useful in the preparation of the [ 18 F]-labelled prosthetic groups. [ 18 F]-labelled compounds useful as radiopharmaceuticals, specifically for use in Positron Emission Tomography (PET) are also provided.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to processes and reagents for[¹⁸F]-fluorination, particularly of peptides. The resultant[¹⁸F]-labelled compounds are useful as radiopharmaceuticals,specifically for use in Positron Emission Tomography (PET).

DESCRIPTION OF RELATED ART

Compounds labelled with short-lived positron emitting radionuclides areused for in vivo studies of human and non-human physiology. Inparticular, radiolabelled bioactive compounds which selectively interactwith specific cell types are useful for the delivery of radioactivity totarget tissues. The applications of bioactive compounds such as peptidesand proteins, including antibodies and fragments of peptides are usefulfor receptor imaging. For example, radiolabelled peptides havesignificant potential for the delivery of radionuclides to the receptorsexpressed on cells of tissues, e.g. in tumours, infarcts and infectedtissues for diagnosis, radiotherapy and monitoring of treatment. PET isa high resolution, non-invasive, imaging technique which has gainedincreased importance in the recent years for the visualisation of humandisease.

In PET, ¹⁸F is one of the most widely used positron-emitting nuclides.¹⁸F is relatively short-lived with a half-life (t_(1/2)) of 110 minutesand is hence a nuclide of choice for receptor imaging. However, one ofthe major disadvantages with [¹⁸F]-labelling of compounds such aspeptides and proteins is the laborious preparation which can only beachieved via prosthetic groups. Bio-molecules cannot be labelleddirectly with fluorine due to the harsh conditions involved. Strategiesfor preparing ¹⁸F-labelled biomolecules require methods where a[¹⁸F]-labelled intermediate is prepared and coupled to the biomolecule,see e.g. WO 99/11590 and references therein.

It is of utmost importance that the preparation of the [¹⁸F]-labelledintermediate and the coupling to the peptide or protein are fast andeasy to perform and that they are amenable to kit formulations for usein the clinical setting. The yield of the preparation of the[¹⁸F]-labelled intermediates ([¹⁸]-labelled prosthetic groups) as wellas the coupling of the intermediate to the peptide or protein should behigh enough so that multiple purifications can be avoided.

Methods for the labelling of biomolecules containing a primary aminegroup such as proteins and peptides with [¹⁸F]-labelled prostheticgroups are continuously sought. Wüst, F., Müller, M. and Bergmann, R. in“Synthesis of 4-([¹⁸]-fluoromethyl)-2-chlorophenylisothiocyanate: Anovel bifunctional ¹⁸F-labelling agent” Radiochim. Acta 92 (2004)349-353, describe a single step synthesis of the ¹⁸F-labelled prostheticgroup of the title and also its conjugation with primary amines. Theauthors however reports that defluorination of the model compoundsoccurred in animal studies.

Therefore, there still exists a need for [¹⁸F]-labelled prostheticgroups and methods which allow rapid introduction of the labelled groupinto biomolecules such as peptides and proteins to give [¹⁸F]-labelledcompounds in high radiochemical yield and purity. Additionally, there isa need for methods which are amenable to facilitate preparations ofradiopharmaceuticals in the clinical setting.

SUMMARY OF THE INVENTION

The present invention provides a process for [¹⁸F]-fluorination ofbiomolecules containing a primary amine group such as proteins andpeptides. The invention further provides reagents for this process, inparticular [¹⁸F]-labelled prosthetic groups for use in the preparationas well as non-labelled intermediates useful in the preparation of the[¹⁸F]-labelled prosthetic groups. [¹⁸F]-labelled compounds useful asradiopharmaceuticals, specifically for use in Positron EmissionTomography (PET) are also provided.

DETAILED DESCRIPTION OF THE INVENTION

The new process and intermediates prepared and used therein; and thecompounds of the invention, their use as radiopharmaceutical imagingagents, their formulation and kits containing them are specified in theattached claims and in the specification hereinafter.

In one embodiment the invention comprises a process for preparingradiofluorinated biomolecules of formula (7)

whereinR¹ denotes H or C₁-C₄ alkyl where methyl is preferred,Spacer denotes a C₁-C₄ alkylene group, optionally interrupted by aoxygen atom and where ethylene is a preferred Spacer,or R¹ and Spacer together with the adjacent nitrogen atom form4-azonia-spiro[3.5]nonyl or 4-aza-4-azonia-spiro[3.5]nonyl groups,R² denotes a bio-molecule residue having at least one free aminofunction, preferably a peptide or protein residue,andX denotes a physiological acceptable anion, preferably a C₁-C₄alkanesulphonate anion, e.g. the methanesulphonate anion,comprisinga) reacting a compound of formula (5)

wherein R¹ and Spacer have the meanings above with a [¹⁸F]-fluoride,andb) reacting the resulting labelled compound of formula (6)

wherein R¹ and Spacer have the meanings abovewith a peptide of formula (8)

R²NH₂  (8)

wherein R² is as defined above,to obtain the labelled [¹⁸F]-fluorinated bio-molecule of formula (7).

The radiofluorination method of step a), useful to prepare the labellingreagent (6), can be carried out by standard methods e.g. in acetonitrilewith [¹⁸F]-KF-Kryptofix at a temperature below 80° C. e.g. by usingmicrowave heating.

After the isolation of compound (6) by cation exchange solid phaseextraction, compound (6) is incubated with a compound of formula R²NH₂(formula 8) in step b) wherein R² is a biomolecule residue to obtain acompound of formula (7).

Isothiocyanates are an established functionality to prepare stablebioconjugates. For examples see: Mao, S.-Y., editor: Walker, John M.“Conjugation of fluorochromes to antibodies” in Protein ProtocolsHandbook (2^(nd) Edition), (2002), 351-354, publisher: Umana Press Inc.and Totowa, N.J.; Jones, N.-., Dive, C. “Cell sensitivity assays:Detection of apoptotic cells in vitro using the TUNEL assay” Methods inMolecular Medicine 28 (1999) 31-38.

The compounds of formula (8) may contain any bio-molecule having a freeprimary amino group. Hence, R² designates the residue of a bio-moleculehaving a free amino function, e.g. a protein or a peptide having a freeamino function at the N-terminal end and/or in the amino acid residue.Preferred such amino acids are lysine, hydroxylysine and arginine.Suitable groups R² comprise proteins, hormones, oligonucleotides,polyclonal and monoclonal antibodies and antibody fragments and peptidemimetics that are useful as targeting vectors.

The present process is particularly attractive for preparing relativelybig peptides where there is no modification and/or control of thelabelling site. The [¹⁸F]-labelling reagent will bind to any availableamino function on a peptide such as lysines or to the N-terminus aminoacid.

Peptide residues comprising somatostatin analogues, such as octreotide,bombesine, vasoactive intestinal peptide, chemotactic peptide analogues,α-melanocyte stimulating hormone, neurotensin, Arg-Gly-Asp peptide andits analogues, human pro-insulin connecting peptide, insulin,endothelin, angiotensin, formyl,norleucyl-phenylalanyl-norleucyl-tyrosyl-lysine and annexin V are usefulclasses of biomolecules in the context of the invention. A further classof peptides for labelling are peptides containing the Arg-Gly-Aspsequence and its analogues, such as those described in WO 01/77145 andWO 03/006491. In one particular aspect, the compound R²NH₂ is of formula(A)

wherein X⁷ has the meaning of

wherein a is an integer of from 1 to 10, preferably a is 1.

The [¹⁸F]-labelled compounds of formula (7) wherein R² are as describedabove, are novel compounds that are useful as radiopharmaceuticals foruse in PET imaging and represent another embodiment of the presentinvention. The radiotracers or formula (7) are sufficiently stable atphysiological pH to allow use as in vivo diagnostic agents. Thestability of the [¹⁸F]-fluoropropyl group has been verified in a numberof examples in vivo, see for instance: Cai, L., Chin, F. T., Pike, V.W., Toyama, H. Liow, J.-S., Zoghbi, S. S. et al. in “Synthesis andEvaluation of Two ¹⁸F-Labeled6-Iodo-2-(4′-N,N-dimethylamino)phenylimidazo[1,2-a]pyridine Derivativesas Prospective Radioligands for Amyloid in Alzheimer's Disease” J. Med.Chem. 47 (2004) 2208-2218 and Maeda, M. Sasaki, S., Fukumura, T. et al.in “Positron-Emitting N—[¹⁸F]Fluoroalkyl and [¹⁸F]FluoropyrrolidinylAnalogues of Eticloprode as potential in Vivo Radioligands for DopamineD₂ Receptors” Chem. Pharm. Bull 40 (1992) 1793-1798.

Hence, compounds of formula (7) comprise a further embodiment of theinvention.

The [¹⁸F]-labelled compounds of formula (6) are novel compounds usefulas intermediates for the preparation of radiopharmaceuticals for use inPET imaging and form a further embodiment of the present invention.

The compounds of formula (5) are also novel compounds useful asintermediates in the preparation of [¹⁸F]-labelled prosthetic groups andform a still further embodiment of the present invention. Thenon-radioactive compound of formula (5) may form part of aradiofluorination kit for the preparation of compounds (6) and (7).

The process further comprises preparing the intermediate compounds offormula (2), formula (3), formula (4) and of formula (5).

c) The compound of formula (2)

whereinA denotes an amino protection group, e.g. BOC (tert-butyl ester), andSpacer and R¹ have the meanings aboveis prepared from a halide of formula (1)

whereinY denotes a halogen atom, e.g. a bromine atom, andSpacer and A have the meanings aboveby reaction with a compound of formula (9)

R¹—NH—CH₂—CH₂—CH₂—OH  (9)

Compounds of formula (1) are commercially available.The compound of formula (1) is reacted with a compound of formula (9),e.g. with 3-aminomethylpropan-1-ol in the presence of potassiumcarbonate and potassium iodide to give an aminoalcohol of formula (2),following the reaction procedure described by Kung, P.-P., Bharadwaj,R., Fraser, A. S., Cook, D. R., Kawasaki, A. M., and Cook, P. in“Solution-Phase Synthesis of Novel Linear Oxyamine CombinatorialLibraries with Antibacterial Activity”, J. Org. Chem. 63 (1998),1846-1852).d) The aniline compounds of formula (3)

wherein R¹ and Spacer have the meanings above, is prepared from thecompound of formula (2) by removal of the protecting group A such as theBOC group, e.g. by using trifluoroacetic acid.e) The isothiocyanate compounds of formula (4)

wherein R¹ and Spacer have the meanings above, are prepared by treatmentof the compound of formula (3) with thiophosgene as outlined for ananalogous compound by Wüst, F., Müller, M. and Bergman, R., in“Synthesis of 4-([¹⁸F]-fluoromethyl)-2-chlorophenylisothiocyanate: Anovel bifunctional ¹⁸F-labelling agent.” Radiochim. Acta 92 (2004),349-353.f) The isothiocyanates of formula (4) are converted into the azetidiniumsalts, such as an azetidium methansulphonate, containing anisothiocyanate function of formula (5)

wherein R¹ and Spacer have the meanings above,

The preparation of the azetidinium salts containing the isothiocyanatefunction can be performed according to the procedure described byKiesewetter, D. O. and Eckelman, W. C. “Utility of azetidiummethanesulfonates for radiosynthesis of 3-[¹⁸F]-fluoropropyl amines” inJ. Label. Compd. Radiopharm. 47 (2004) 953-969 for the corresponding7-[(4-cyanophenoxy)methyl]-4-azoniaspiro[3.5]nonane methanesulphonate.

The preparation is further illustrated in Scheme 1 wherein in thegeneral formulas (1) to (7) the substituents have the followingmeanings:

R¹ denotes a methyl groupR² denotes RA denotes BOCY denotes BromineSpacer denotes ethylene

The ¹⁸F-labelled compounds of formula (7) are useful asradiopharmaceutical imaging agents, specifically as imaging agents foruse in Positron Emission Tomography (PET). For use as imaging agents thecompounds of formula (7) are preferably formulated as imaging agentcompositions with solvents and/or excipients known from the state ofart.

In a still further embodiment the invention comprises novel chemicalcompounds that are useful e.g. as intermediates in the process specifiedabove and being of the formulas (2), (3) and (4):

wherein R¹ and Spacer have the meanings specified above

wherein R¹ has the meanings specified above and Spacer denotes a C₂-C₄alkylene group, optionally interrupted by a oxygen atom, or R¹ andSpacer together with the adjacent nitrogen atom form4-azonia-spiro[3.5]nonyl or 4-aza-4-azonia-spiro[3.5]nonyl groups,

wherein R¹ has the meanings above, Spacer denotes a C₂-C₄ alkylenegroup, optionally interrupted by a oxygen atom or R¹ and Spacer togetherwith the adjacent nitrogen atom form 4-azonia-spiro[3.5]nonyl or4-aza-4-azonia-spiro[3.5]nonyl groups, and A denotes an amino protectiongroup such as BOC (tert-butyl ester).

In a method of diagnosis, compounds of formula (7) preferably providedas compositions as devised above, are administered to a human or animalbody. Alternatively, the human or animal body is pre-administered withthe compound of formula (7) prior to the commencement of the method ofdiagnosis. The body is examined with a diagnostic device and data arecompiled from the examination. In an additional step the data may be areanalysed to reach to a diagnosis. Preferably the compounds of formula(7) are used in PET imaging, where the diagnostic device is a PositronEmission Tomography scanner.

The invention further comprises a radiofluorination kit comprising acompound of formula (5) for the preparation of compounds of formulas (6)and (7). The radiofluorination kit may further comprise a compound offormula (8) and necessary additional reagents for the preparation of theimaging agent of formula (7).

EXAMPLES Example 1 Preparation of compound(2)—(4-{2-[(3-Hydroxy-propyl)-methyl-amino]-ethyl}-phenyl)-carbamic acidtert-butyl ester (step c)

One equivalent of compound (1), 0.5 equivalents of potassium carbonate,and 0.01 equivalents of potassium iodide are added sequentially to asolution of 3-methylaminopropan-1-ol (one equivalent) in DMF. Theresulting mixture is stirred at 65° C. for 12 h. The reaction mixture isevaporated in vacuo and partitioned between ethyl acetate and water. Thecombined organic layers are dried (Na₂SO₄), filtered, and evaporated togive a residue. Purification of the residue by flash columnchromatography produces the compound of formula (2).

Example 2 Preparation of compound(3)—(3-{[2-(4-Amino-phenyl)-ethyl]-methyl-amino}-propan-1-ol (step d)

Compound (2) is dissolved in dichloromethane and trifluoroacetic acid(10 equivalents) added with stirring. After stirring for two hours atroom temperature, the solvent is removed in vacuo. The residue isdissolved in dichloromethane and washed with a 5% sodium bicarbonatesolution, dried with brine and magnesium(II) sulfate. Removal of thesolvent affords compound (3).

Example 3 Compound(4)—3-{[2-(4-Isothiocyanato-phenyl)-ethyl]-methyl-amino}-propan-1-ol(step e)

To a suspension of compound (3) with barium(II) carbonate (5equivalents) in chloroform a solution of thiophosgene (one equivalent)in chloroform is added at 0° C. with stirring. After warming up to roomtemperature, stirring is continued for one hour. Water is added and thecrude product extracted with chloroform. After drying with brine andmagnesium(II) sulfate, the product is purified by flash chromatography(silica, ethyl acetate/50% n-hexane).

Example 4 Compound (5)—Methanesulfonate1-[2-(4-isothiocyanato-phenyl)-ethyl]-1-methyl-azetidinium (step f)

A solution of compound (4) in chloroform is mixed with methanesulfonylchloride (1.1 equivalents) and triisopropylethylamine (4 equivalents)and stirred for one hour at room temperature. The intermediate mesylateis isolated by flash chromatography (silica, ethyl acetate/50% n-hexane)and subsequently refluxed in chloroform for 12 h. The solvent is removedin vacuo and the residue triturated with ethyl acetate to form compound(5) as a solid.

Example 5 Compound(6)—(3-[¹⁸F]-Fluoro-propyl)-[2-(4-isothiocyanato-phenyl)-ethyl]-methyl-amine(step a)

To a Wheaton vial charged with Kryptofix® (10 mg), potassium carbonate(1 mg dissolved in 0.05 ml water), and acetonitrile (0.8 ml) thefluorine-18 containing water (10 mCi, 1 ml) is added. The solvent isremoved by heating at 110° C. for 30 min under a stream of nitrogen.Anhydrous acetonitrile (0.5 ml) is added and again evaporated as before.This step is repeated twice. The vial is cooled to room temperaturefollowed by injecting a solution of compound (5) (1 mg) in anhydrousacetonitrile (0.2 ml). The reaction mixture is heated at 80° C. for 5min and quenched in 0.5 ml of water. Compound (6) is isolated by solidphase extraction from an SCX cartridge (washing with water and elutingproduct by using a solution of water/25 ethanol).

Example 6 Compound (7)—Radiolabelling Annexin-V with Compound (6) (Stepb)

A solution of compound (6) is concentrated by a stream of nitrogen to0.05 ml. A solution of annexin-V in borate buffer pH 8.0 (0.1 M) isprepared by dialysis and mixed with compound (6). After incubation for30 min at room temperature, the ¹⁸F-labelled annexin-V (7) is purifiedusing size exclusion chromatography.

What is claimed is: 1-26. (canceled)
 27. Radiopharmaceutical compositioncomprising a compound of formula (7)

wherein R¹ denotes H or C₁-C₄ alkyl, Spacer denotes a C₁-C₄ alkylenegroup optionally interrupted by an oxygen atom, R² denotes abio-molecule residue having at least one free amino function, and Xdenotes a physiological acceptable anion, optionally together with asolvent or excipient.
 28. Compound of formula (6)

wherein R¹ denotes H or C₁-C₄ alkyl, Spacer denotes a C₁-C₄ alkylenegroup optionally interrupted by an oxygen atom, and X denotes aphysiological acceptable anion.
 29. Compound of formula (4)

wherein R¹ denotes H or C₁-C₄ alkyl and Spacer denotes a C₁-C₄ alkylenegroup optionally interrupted by an oxygen atom.
 30. Compound of formula(3):

wherein R¹ denotes H or C₁-C₄ alkyl and Spacer denotes a C₁-C₄ alkylenegroup optionally interrupted by an oxygen atom.
 31. Compound of formula(2):

wherein R¹ denotes H or C₁-C₄ alkyl, Spacer denotes a C₁-C₄ alkylenegroup optionally interrupted by an oxygen atom, and A denotes an aminoprotection group such as BOC (tert-butyl ester).
 32. Compound of formula(5)

wherein R¹ denotes H or C₁-C₄ alkyl, Spacer denotes a C₁-C₄ alkylenegroup optionally interrupted by an oxygen atom, or R¹ and Spacertogether with the adjacent nitrogen atom forms a4-azonia-spiro[3.5]nonyl group, and X denotes a physiological acceptableanion.
 33. Radiofluorination kit comprising a compound (5):

wherein R¹, Spacer, and X have the meanings of claim
 35. 34.Radiofluorination kit of claim 36 further comprising a compound offormula (8):R²NH₂  (8) wherein R² denotes a bio-molecule residue having at least onefree amino function.